US11808702B2 - Hybrid multi-photon microscopy - Google Patents

Hybrid multi-photon microscopy Download PDF

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US11808702B2
US11808702B2 US17/427,586 US202017427586A US11808702B2 US 11808702 B2 US11808702 B2 US 11808702B2 US 202017427586 A US202017427586 A US 202017427586A US 11808702 B2 US11808702 B2 US 11808702B2
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photon
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Alipasha Vaziri
Siegfried Weisenburger
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Rockefeller University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B9/00—Measuring instruments characterised by the use of optical techniques
    • G01B9/04—Measuring microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/061—Sources
    • G01N2201/06113—Coherent sources; lasers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B2207/00—Coding scheme for general features or characteristics of optical elements and systems of subclass G02B, but not including elements and systems which would be classified in G02B6/00 and subgroups
    • G02B2207/114—Two photon or multiphoton effect

Definitions

  • a major goal of modern neuroscience is to understand how neural networks perform cognitively relevant functions. In order to achieve this goal, it is useful to simultaneously and independently record activities of large neuronal populations that are building blocks of even the simplest neural networks. This task has been hampered, however, by shortcomings in available tools and technologies.
  • Information related to sensory inputs, behavioral states, and cognitive functions are all thought to be represented on multiple spatial and temporal scales in a distributed fashion and over large neuronal networks that can span up to the level of entire brains.
  • the ability to capture the dynamics of such large neuronal ensembles across multiple regions of the brain at sufficient spatial and temporal resolution is thus essential in order to understand how various cognitive functions and complex behavior emerge from the activity of neuronal populations.
  • an integrated systems-wide optimization approach is provided in combination with multiple technical innovations, wherein a new design paradigm for optical microscopy that is based on maximization of biological information content as measured by the fidelity of obtained neuronal signals is presented.
  • a modular design utilizes a hybrid multi-photon acquisition approach and allows volumetric recording of neuroactivity at single-cell resolution within volumes of up to 1,000 ⁇ 1,000 ⁇ 1,220 ⁇ m at up to 17 Hz in awake behaving mice.
  • a multi-photon imaging system includes a laser module having a first channel for outputting a two-photon excitation laser pulse and a second channel for outputting a three-photon excitation laser pulse.
  • the system further includes a first optical path for guiding the two-photon laser pulse from the first channel of the laser module and a second optical path for guiding the three-photon laser pulse from the second channel of the laser module.
  • a microscope is also provided for simultaneously receiving the two-photon laser pulse from the first optical path and the three-photon laser pulse from the second optical path, and simultaneously, or with well controllable delays, delivering the two-photon laser pulse and the three-photon pulse to a target volume.
  • the system further includes a photodetector configured to collect photons generated within the target volume in response to simultaneous excitation of the target volume by both the two-photon laser pulse and the three-photon laser pulse.
  • the first optical path includes a multiplexing module configured to split the two-photon excitation laser pulse received from the laser module into a plurality of sub-pulses including a first sub-pulse and a second sub-pulse.
  • the multiplexing module is further configured to introduce a time delay between the first sub-pulse and the second sub-pulse.
  • the first optical path may also include a temporal focusing module having an optical grating for spatially dispersing the spectrum of at least one of the two-photon excitation laser pulse or the three-photon excitation laser pulse.
  • the first optical path may also include a remote scanning module having a mirror movable in an axial direction of the first optical path for axially scanning the two-photon excitation laser pulse between different depths within the target volume.
  • the second optical path may also include a remote scanning module including a mirror movable in an axial direction of the second optical path for axially scanning the three-photon excitation laser pulse between different depths within the target volume.
  • the system may further include an actuator for moving the microscope in a direction of the received two-photon laser pulse and the received three-photon laser pulse for axially scanning the two-photon laser pulse and the three-photon laser pulse between different depths within the target volume.
  • Both the first optical path and the second optical path may include a two-dimensional lateral scanning module for respectively angularly deflecting the two-photon laser pulse and the three-photon laser pulse, whereby the two-photon laser pulse and the three-photon laser pulse are respectively scanned on first and second axial planes within the target volume.
  • An imaging system may include a laser module for outputting a laser pulse, a multiplexing module configured to split the laser pulse received from the laser module into a plurality of sub-pulses including a first sub-pulse and a second sub-pulse and to introduce a time delay between the first sub-pulse and the second sub-pulse, a temporal focusing module including an optical grating for spatially dispersing the spectrum of the first sub-pulse and the second sub-pulse, a remote scanning module for receiving the first sub-pulse and the second sub-pulse and including a mirror movable in an axial direction of the first sub-pulse and the second sub-pulse for axially scanning the first sub-pulse and the second sub-pulse between different depths within the target volume, a telescope for receiving the first sub-pulse and the second sub-pulse from the remote scanning module and delivering the first sub-pulse and the second sub-pulse
  • the imaging system may further include a lateral scanning module for respectively angularly deflecting the first sub-pulse and the second sub-pulse, whereby the first sub-pulse and the second sub-pulse are respectively scanned on first and second axial planes within the target volume.
  • a lateral scanning module for respectively angularly deflecting the first sub-pulse and the second sub-pulse, whereby the first sub-pulse and the second sub-pulse are respectively scanned on first and second axial planes within the target volume.
  • a method for multi-photon imaging of fluorophores within a target volume includes providing a two-photon laser pulse from a laser module, providing a three-photon laser pulse from the laser module, guiding the two-photon laser pulse from the laser module along a first optical path, guiding the three-photon laser pulse from the laser module along a second optical path, simultaneously delivering the two-photon laser pulse from the first optical path and the three-photon laser pulse from the second optical path to a microscope, simultaneously delivering the two-photon laser pulse and the three-photon pulse from the telescope to a target volume and simultaneously collecting photons generated within the target volume in response to simultaneous excitation of the target volume by both the two-photon laser pulse and the three-photon laser pulse with a photodetector.
  • the method may further include splitting the two-photon pulse into a plurality of sub-pulses including at least a first sub-pulse and a second sub-pulse and introducing a time delay between the first sub-pulse entering the target volume and the second sub-pulse entering the target volume.
  • the method may further include spatially dispersing the spectrum of the two-photon laser pulse.
  • the method may further include axially scanning the two-photon laser pulse between different depths within the target volume.
  • the method may further include simultaneously axially scanning the two-photon laser pulse and the three-photon laser pulse between different depths within the target volume.
  • the method may further include angularly deflecting the two-photon laser pulse and the three-photon laser pulse, whereby the two-photon laser pulse and the three-photon laser pulse are respectively scanned on first and second axial planes within the target volume.
  • a method for high-speed imaging of fluorophores within a target volume includes providing a pulsed laser beam from a laser module, splitting the pulsed laser beam from the laser module into a plurality of sub-pulses with a multiplexing module such that the plurality of sub-pulses includes a first sub-pulse and a second sub-pulse, introducing a time delay between the first sub-pulse and the second sub-pulse, spatially dispersing the spectrum of the first sub-pulse and the second sub-pulse, axially scanning the first sub-pulse and the second sub-pulse between different depths within the target volume, delivering the first sub-pulse and the second sub-pulse to a target volume and collecting photons generated within the target volume in response to simultaneous excitation of the target volume by both the first sub-pulse and the second sub-pulse.
  • the method may further include respectively angularly deflecting the first sub-pulse and the second sub-pulse, whereby the first sub-pulse and the second sub-pulse are respectively scanned on first and second axial planes within the target volume.
  • FIG. 1 is a schematic diagram of a 2p multiplexing temporal focusing (MuST) microscope with remote scanning, according to the present invention.
  • FIG. 2 is a schematic diagram of a remote scanning module, according to the present invention.
  • FIG. 3 is a schematic diagram illustrating an arrangement of multiplexed temporal focusing (MuST) spots for the 4 ⁇ axial multiplexing and the 4 ⁇ lateral multiplexing configuration of the 2p-MuST microscope.
  • MuST multiplexed temporal focusing
  • FIG. 4 is a perspective view of a mirror holder chuck showing the beamlet arrangement for 4 ⁇ axial multiplexing, according to the present invention.
  • FIG. 5 is a schematic diagram showing the overall hybrid 3p/2p-MuST microscope and data acquisition system of the present invention.
  • FIG. 6 is a schematic diagram illustrating light sculpting using a temporal focusing (TeFo) module according to the present invention.
  • One of the goals of the present invention microscope design was to faithfully extract neuronal activity at the precision of single neuronal cell bodies, ⁇ 10-15 ⁇ m, from target volumes that span ⁇ 1 mm axially and ⁇ 1 mm laterally at a minimum volume rate set by the time scale of typical Ca 2+ transients while maintaining the sample exposure to laser power within established safe limits.
  • the voxel size is on the order of the diffraction-limited point spread function (PSF) size ( ⁇ 0.5 ⁇ 0.5 ⁇ 2 ⁇ m 3 ) which together with the typical sample-to-sample spacing of ⁇ 0.5 ⁇ m results in slow imaging rates when the aim is to record from a large volume. For example, a 1,000 ⁇ 1,000 ⁇ 600 ⁇ m volume would be composed of ⁇ 1.2 ⁇ 10 9 voxels.
  • PSF point spread function
  • the volume acquisition rates using a typical 80-MHz repetition rate laser would be in the range of ⁇ 0.1 Hz, even when assuming no additional limitations due to the scan hardware, thus insufficient to capture neuronal dynamics distributed across the volume.
  • the lateral size d of excitation in a diffraction-limited spot can resolve anatomical features that are on the order of d ⁇ /(2NA) ⁇ 0.5 ⁇ m
  • the localization of a Gaussian beam along the axial direction z is proportional to ⁇ d 2 , thus larger, and intrinsically coupled to the lateral localization of excitation. Therefore, by decoupling the lateral from the axial confinement of excitation, one could in principle generate a laterally larger but more isotropically shaped PSF that would more adequately sample the volume along all dimensions when the ⁇ 0.5 ⁇ m lateral resolution of diffraction-limited scanning is not required.
  • a nearly isotropically shaped 5- ⁇ m spot size is used, together with a 5- ⁇ m sampling of a target volume of ⁇ 1 mm 3 .
  • light sculpting based on temporal focusing is utilized.
  • TeFo the spectrum of an ultrafast laser is typically spatially dispersed by a grating and imaged into the sample. This arrangement results in a geometric dispersion of the pulse frequency components everywhere but in one focal plane within the sample, achieving an axial confinement of the excitation that can be controlled by the spectral filling of the microscope objective back focal aperture.
  • the present invention utilizes a custom laser system for this wavelength that could provide the highest possible repetition rate while maintaining a sufficient pulse energy of >100 nJ to excite larger PSFs with sufficient SNR.
  • the system of the present invention consists of a fiber chirped pulse amplifier (FCPA) that pumps an optical parametric chirped pulse amplifier (OPCPA).
  • FCPA fiber chirped pulse amplifier
  • OPCPA optical parametric chirped pulse amplifier
  • the OPCPA has an output at ⁇ 960 nm with a repetition rate of ⁇ 4.7 MHz while providing >800 nJ, sufficient for a one-pulse-per-voxel acquisition scheme. This acquisition scheme allows for the maximization of the obtainable fluorescence signal and SNR from each voxel for the lowest possible single-photon induced heat penalty.
  • the present invention combines light-sculpted PSF with spatiotemporal multiplexing resulting in a Multiplexed Scanned Temporal Focusing (MuST) microscope.
  • the excitation beam 10 is split into four beamlets 10 a , 10 b . 10 c . 10 d and sent through a delay stage that introduces a temporal delay of ⁇ 8 ns between each of the four beamlets, as shown in FIG. 1 .
  • FIG. 1 shows a multiplexing module 30 creating four beamlets using PBS (polarizing beam splitters) and HWP (half wave plates), each delayed by 8 ns, for example, with respect to the adjacent one.
  • PBS polarizing beam splitters
  • HWP half wave plates
  • the required temporal delay between the beamlets is determined by the combined effects of the fluorophore's fluorescent lifetime, which is ⁇ 2.7 ns for GCaMP Ca 2+ indicators, convolved with the instrument response function of the data acquisition driven mainly by the detector and digitization bandwidths.
  • the chosen 8-ns delay between the beamlets allows de-multiplexing of the fluorescent signals from subsequent beamlets with negligible cross-talk.
  • this scheme allows for an effective time-tagging of the four excitation spots such that their fluorescence signals could be discriminated by a single PMT based on their different detection times.
  • Tissue heating due to linear absorption is the main known damage mechanism in nonlinear microscopy and it has been shown that average power levels above ⁇ 250 mW can lead to heat-induced tissue damage as reported via immunohistochemical markers.
  • the tolerable levels of average power impose a limitation on the pulse energy at the effective repetition rate and for the larger, light-sculpted PSF, while using a single-pulse-per-voxel excitation scheme results in an ⁇ ntimes SNR gain for the same average power (compared to averaging n laser pulses).
  • a model is presented that accounts for the various system parameters such as collection efficiency or digitizer dynamic range as well as the laser spot size and shape, and the characteristics of the Ca 2+ indicators.
  • the sensitivity, S the true-positive rate of identifying neuronal signals against a ground truth, is used as the figure of merit in the signal extraction pipeline. While dependent on SNR—as well as the spatial resolution and acquisition rate—the sensitivity (S), is the better figure of merit, as it allows for direct optimization of the microscope based on a key performance factor that is of ultimate biological relevance. Thereby the minimally required laser pulse energy resulting in a sufficient SNR—but not necessarily the highest possible—for high-fidelity neuronal signal extraction could be identified.
  • a high sensitivity (S>0.9) can be achieved using a 5- ⁇ m TeFo spot and a combined pulse energy of ⁇ 10-20 nJ for all four excitation spots, corresponding to a total average power of ⁇ 50-100 mW while maintaining high fidelity in neuronal signal detection.
  • the diffraction-limited PSF under the same conditions only reaches a sensitivity of S ⁇ 0.75 due to under-sampling.
  • Increasing the lateral size of the PSF requires higher pulse energies to maintain the same signal level. However, this is not the case for the axial extent of the PSF because the fluorescence signal is proportional to the photon flux through the excitation area. Thus, while from a purely spatial sampling perspective an isotropically shaped PSF would be desirable, optimization of the axial size of the PSF under maximization of S and volume acquisition speed results in a larger axial PSF size up to the limit of the cell body diameter (10-15 ⁇ m) beyond which decrease in SNR and increased crosstalk reduce.
  • An optimum lateral 2p excitation spot size of ⁇ 5 ⁇ m and an axial size of ⁇ 10-15 ⁇ m can be used as target parameters for sculpted 2p-MuST PSF.
  • scanning hardware for in-plane imaging can easily achieve sufficient frame rates for Ca 2+ imaging using fast resonant scanners ( ⁇ 16 kHz bi-directional rate) and galvo scanners (>1 kHz line rate), scanning in the axial (z) direction is typically slower and more challenging.
  • Axial scanning of the microscope objective using a piezoelectric element is inertia-limited and thus slow when using a long translation range of ⁇ 0.5-1 mm. Moreover, it could cause aberrations and vibrations that are transferred to the sample.
  • FIG. 2 shows a remote scanning module 40 according to one aspect of the present invention.
  • the remote scanning module 40 includes a polarizing beam splitter (PBS) 42 ), a quarter wave plate (QWP) 44 and at least one mirror (M) 12 mounted on a voice coil actuator (VC) 14 that can modulate the beam divergence for axial z-scanning.
  • PBS polarizing beam splitter
  • QWP quarter wave plate
  • M mirror
  • VC voice coil actuator
  • four (4) mirrors 12 are preferably mounted on the voice coil actuator 14 for respectively reflecting the four beamlets 10 a , 10 b . 10 c . 10 d received from the multiplexing module 30 .
  • TeFo is combined with remote axial scanning by inserting the axially movable mirror 12 into a plane conjugated to both the grating plane and the sample plane.
  • Axial scanning of the mirror 12 results in a modulation of the beam divergence of the reflected beam, which results in a shift of the axial position of the light-sculpted TeFo spot.
  • the laser pulses are spatially dispersed before the remote scanning module, the geometric focal plane and the temporal focusing plane coincide for all remote scanning z-positions. Thus, the axial confinement characteristics of the excitation as provided by temporal focusing are not affected.
  • FIG. 3 illustrates an arrangement of multiplexed temporal focusing (MuST) spots for the 4 ⁇ axial multiplexing and the 4 ⁇ lateral multiplexing configuration of the 2p-MuST microscope.
  • MuST multiplexed temporal focusing
  • a microscope is configured for a 4 ⁇ axial or 4 ⁇ lateral multiplexing scheme by designing different mirror holder chucks that were attached to the voice coil actuator.
  • the design of the present invention is fully modular and as such it also allows to use any of the modules individually or in combination. For example, a remotely scanned temporal focusing module can be readily added to an existing 2p-scanning microscope. Overall, this design allowed for a voxel acquisition rate of 18.8 MHz covering up to 0.6 mm 3 in its different configurations.
  • each of the four beamlets will cover an axial range of 150 ⁇ m in their respective sub-volumes in the sample.
  • a specially designed mirror holder chuck 16 for the voice coil actuator 14 is provided, as shown in FIG. 4 .
  • the chuck 16 includes a base plate 18 for mounting to the voice coil actuator 14 .
  • the base plate 18 supports and allows for the positioning of four small mirrors 12 at different z-positions such that an axial separation of ⁇ 150 ⁇ m for the four beamlets could be achieved in the sample. Since the spots of the four beamlets on the mirrors 12 are laterally separated, the four sub-volumes in the sample also show a lateral displacement. By careful alignment of the beamlets, this lateral displacement of the sub-volumes can be minimized to less than ⁇ 95 ⁇ m.
  • the microscope for 4 ⁇ lateral multiplexing. This is accomplished by aligning the four spatiotemporally multiplexed beamlets laterally using a different mirror holder chuck on the voice coil actuator such that each beamlet covered laterally a quarter of the target FOV in the sample.
  • FIG. 5 shows a hybrid multi-photon microscope system 20 according to the present invention.
  • the system 20 combines a 2p-MuST optical path 22 with a 3p excitation path 23 into a multiphoton hybrid microscope.
  • the present invention integrates the 3p excitation beam path 23 as the fifth beamlet, which can be delayed by ⁇ 8 ns with respect to the 2p beamlets. This is possible because of a specially designed laser system where both OPCPA channels 26 are pumped by the same FCPA laser 28 , ensuring that the relative pulse delays are maintained.
  • This synchronization of the pulse trains from the two OPCPA arms 26 a , 26 b allows the use of the same PMT and data acquisition pipeline 29 to detect the fluorescence signal from all five (four 2p-MuST and one 3p) excitation spots.
  • the system 20 preferably includes a custom laser system consisting of an Yb-fiber chirped pulse amplifier (FCPA) 28 and an optical parametric chirped pulse amplifier (OPCPA) 26 .
  • the OPCPA 26 has two output channels 26 a , 26 b at 960 nm and 1,300 nm wavelength for 2p and 3p excitation, respectively.
  • the 960 nm channel 26 a produces >0.8- ⁇ J, ⁇ 90-fs pulses at a repetition rate of 4.7 MHz.
  • the 1,300 nm channel 26 b produces >1.4- ⁇ J, ⁇ 70-fs pulses at a repetition rate of 1.0 MHz.
  • Both channels are preferably dispersion compensated in respective dispersion compensation modules 54 , 55 which may include custom chirped mirror pairs (960 nm channel) or a prism compressor (1,300 nm channel).
  • the 2p beam is split into four beamlets in the spatiotemporal multiplexing module 30 .
  • the spatiotemporal multiplexing module 30 uses polarizing beam splitters (PBS) 56 with half-wave plates (HWPs) for power adjustment of each beamlet.
  • PBS polarizing beam splitters
  • HWPs half-wave plates
  • the beamlets can be delayed by 8, 16 and 24 ns, for example, using beam paths with relative length differences of ⁇ 2.5, ⁇ 5 and ⁇ 7.5 m, respectively.
  • two additional HWPs are used whereby that the polarization can be adjusted such that all four beamlets are TE polarized.
  • the beam diameters of each beamlet 10 a , 10 b , 10 c , 10 d are adjusted using individual telescopes, and the beams were then converged onto a plane that is conjugated to the temporal focusing module 50 .
  • Light sculpting of the 2p beam is preferably achieved with a temporal focusing (TeFo) module 50 .
  • TeFo temporal focusing
  • the spectrum of an ultrafast laser is spatially dispersed by a grating 52 and imaged into the sample plane.
  • This arrangement results in a geometric dispersion of the pulse frequency components everywhere but in the focal plane within the sample, achieving an axial confinement of the excitation that can be controlled by the spectral filling of the microscope objective back focal plane (BFP). This allows an effective decoupling of the lateral confinement from the axial confinement of the excitation spot.
  • the temporal focusing module 50 includes a temporal focusing lens and a D-shaped mirror.
  • the four beamlets 10 a , 10 b , 10 c , 10 d are directed to the temporal focusing lens by the D-shaped mirror at a slight vertical angle.
  • the beamlets are then focused onto two small holographic diffraction gratings 52 arranged in a Littrow configuration.
  • the first diffraction order is spatially dispersed in the horizontal axis and collimated using the same lens before it is directed to the remote scanning module 40 .
  • the remote scanning module 40 includes a polarizing beam splitter (PBS) 42 , a quarter wave plate (QWP) 44 and a mirror (M) arrangement 12 mounted on a voice coil actuator (VC) 14 .
  • the mirror arrangement 12 includes a mirror holder chuck 16 that supports four small (5 ⁇ 5 ⁇ 2 mm) mirrors 12 a , 12 b , 12 c , 12 d at respectively different axial positions along the beam path.
  • the beamlets 10 a , 10 b , 10 c , 10 d are directed to different positions in the sample.
  • the mirror holder chuck 16 is mounted on a fast voice coil actuator 14 , which allows rapid remote scanning to continuously acquire volumes by saw-tooth axial z-scanning.
  • the same lens 58 re-collimates the beamlets 10 a , 10 b , 10 c , 10 d and after the second pass through the QWP 44 , the PBS 42 directs the beamlets to the microscope.
  • a high-speed driver is used to directs the beamlets to the microscope.
  • the remote scanning module 40 After passing through independent sets of galvo mirrors 32 , 33 and resonant scanners 34 , 35 , which are preferably phase-locked to each other in a master/slave configuration, the 2p and 3p beam paths are combined using a dichroic mirror 36 , while axial scanning in this case was performed using a microscope objective piezo 38 .
  • the remote scanning module 40 (as also shown in FIG. 2 ), preferably utilizes an adjustable mirror holder mount. The remote scanning module 40 enables free selection of the axial positions of the four 2p-MuST sub-volumes with respect to each other and with respect to the 3p volume which was kept at the native focal plane. This allows for the adjustment of the size and geometry of the imaged volume to regions of interest in the sample.
  • the microscope setup 38 preferably includes an identical galvo/resonant scanning (15.5 kHz) mirror pair for both the two-photon and three-photon paths.
  • the two resonant scanners 34 , 35 are controlled in a phased-locked master/slave configuration.
  • the scan and relay lenses are preferably chosen such that the 2p scanned temporal focusing spot on the grating is imaged into the sample exciting a volume of 5 ⁇ 5 ⁇ 15 ⁇ m and that the 3p spot excites a volume of 1.5 ⁇ 1.5 ⁇ 9.4 ⁇ m, respectively.
  • the 960 nm and 1,300 nm channels are combined before the tube lens using a custom low-dispersion dichroic.
  • a 0.8 NA, 16 ⁇ water-immersion, long working distance microscope objective is used.
  • the objective is preferably mounted on a long travel range (1 mm), high-speed piezo stage, which can be used for 3 photon and hybrid recordings to continuously acquire volumes by saw-tooth axial z-scanning of the microscope objective.
  • the piezo uses a high-speed driver which allows for an optimized flyback and settling time of ⁇ 20 ms over a ⁇ 150-250 ⁇ m travel range.
  • the resulting signal is collected using wide-angle optics, and detection is performed using a photon multiplier tube 29 .
  • a data acquisition scheme, according to the present invention, is also shown in FIG. 5 .
  • the PMT signal is preferably recorded by an 800-MHz digitizer.
  • a field programmable gate array (FPGA) 58 allows for synchronization of the voxel acquisition to the pulse repetition rate of the laser and to realize a scheme in which each voxel is excited only by a single laser pulse.
  • the FPGA clock at 750 MHz is preferably provided by an evaluation board and phase-locked to the FCPA oscillator frequency.
  • the FPGA digitizer board is triggered by individual laser pulses from the 960 nm channel at 4.7 MHz and the signal is collected from the PMT within 5 of the 1.33 ns samples.
  • the line synchronization is provided by the master resonant scanner.
  • the laser intensity can be blanked during the turnaround of the resonant scanner and axial flyback time and modulated with increasing image depth.
  • the system of the present invention provides dispersion compensation, multiplexing, temporal focusing and remote focusing modules.
  • the system defines a 2-photon path 22 and a 3-photon path 23 .
  • the data acquisition scheme preferably includes a phase-locked loop (PLL), an analog/digital converter (ADC) a photomultiplier tube (PMT) and a field programmable gate array (FPGA).
  • PLL phase-locked loop
  • ADC analog/digital converter
  • PMT photomultiplier tube
  • FPGA field programmable gate array

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